2.1. THE NOTCH GENE AND PROTEIN
In Drosophila melanogaster, the so called "Notch group" of genes has been implicated in events crucial for the correct developmental choices of a wide variety of precursor cells (for review, see Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408). The accumulated genetic and molecular studies suggest that these genes encode elements of a cell communication mechanism which includes cell surface, cytoplasmic, and nuclear components.
The central player of the Notch group is the Notch (N) locus which encodes a transmembrane protein containing EGF-like repeats in its extracellular domain (Wharton et al., 1985, Cell 43:567-581; Kidd et at., 1986, Mol. Cell. Biol. 6:3094-3108). This protein has been shown to interact molecularly and genetically with two other transmembrane, EGF-containing proteins of the Notch group: Serrate and Delta (Vaessin et al., 1985, J. Neurogenetics 2:291-308; Fehon et al., 1990, Cell 61:523-534; Fleming et al., 1990, Genes Dev. 4:2188-2201; Xu et al., 1990, Genes Dev. 4:464-475; Rebay et al., 1991, Cell 67:687-699; Thomas et al., 1991, Development 111:749-761). The other members of the Notch group are deltex (Xu and Artavanis-Tsakonas, 1991, Genetics 126:665-677), Enhancer of (split) [E(spl)] (Knust et al., 1987, EMBO J. 6:4113-4123; Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927; Klambt et al., 1989, EMBO J. 8:203-210), and mastermind (mam) (Smoller et al., 1990, Genes Dev. 4:1688-1700). mastermind and Enhancer of (split) encode nuclear proteins (Smoller et al., 1990, Genes Dev. 4:1688-1700; Delidakis et al., 1991, Genetics 129:803-823).
Notch homologs have been isolated from a variety of vertebrate species and have been shown to be remarkably similar to their Drosophila counterpart in terms of structure, expression pattern and ligand binding properties (Rebay et al., 1991, Cell 67:687-699; Coffman et al., 1990, Science 249:1438-1441; Ellisen et al, 1991, Cell 66:649-661; Weinmaster et al., 1991, Development 113:199-205). A human Notch (TAN-1) malfunction has been associated with a lymphatic cancer (Ellisen et al, 1991, Cell 66:649-661).
Notch is expressed on axonal processes during the outgrowth of embryonic neurons (Johansen et at., 1989, J. Cell Biol. 109, 2427-2440; Kidd et al., 1989, Genes Dev. 3, 1113-1129). A study has shown that certain Ax alleles of Notch can severely alter axon pathfinding during sensory neural outgrowth in the imaginal discs, although it is not yet known whether aberrant Notch expression in the axon itself or the epithelium along which it grows is responsible for this defect (Palka et at., 1990, Development 109, 167-175).
E(spl) is a complex locus comprised of at least ten genetically related transcription units which have been separated into two distinct groups, both of which display genetic interactions with specific Notch mutations (Knust et al., 1987, EMBO J. 6:4113-4123; Hartley et al., 1988, Cell 55:785-795; Preiss et at., 1988, EMBO J. 7:3917-3927; Klambt et al., 1989, EMBO J. 8:203-210; Delidakis et al., 1991, Genetics 129:803-823). The first group codes for proteins containing the helix-loop-helix motif (Klambt et al., 1989, EMBO J. 8:203-210) while the second displays homology to the .beta. subunit of transducin (Hartley et al., 1988, Cell 55:785-795). Knust et al. (1987, EMBO J. 6:4113-4123) have numbered the transcripts in the E(spl) region and, according to their numbering system, the transcripts coding for the transducin-homologous protein are termed m9/10. Several embryonic lethal alleles affecting this gene were isolated. Moreover, P element transformation analyses demonstrated that the mutation groucho, which affects bristle development in Drosophila, is allelic to the Enhancer of split m9/10 gene (Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927).
The 719 amino acid long product of the E(spl) m9/10 gene contains four tandemly arranged repeats spanning the carboxyl-terminal .about.300 amino acid residues of the protein (Hartley et al., 1988, Cell 55:785-795). Each repeat is approximately 40 residues in length and is characterized by the presence of the conserved motif WDL. Such repeats are found similarly arranged in the .beta. subunits of G proteins and have been referred to as the "WD-40 repeat" (for review, see Simon et al., 1991, Science 252:802-808). Several other proteins containing this structural motif include the products of the yeast cell cycle gene CDC 4 (Yochem and Byers, 1987, J. Mol. Biol. 195:233-245) and of the TUP1 gene, a mediator of glucose repression (Williams and Trumbly, 1990, Mol. Cell. Biol. 10:6500-6511.).
Very little is known about the mechanisms underlying cell fate choices in higher organisms such as vertebrates; a knowledge of such mechanisms could provide insights into pathologies associated with abnormal differentiation events. Thus, a need exists in the art to obtain and characterize the human members of the "Notch group" of genes, including Enhancer of split, since these genes appear to play crucial roles in the determination of cell fate.